Method for producing beta-hydroxy amino acid and enzyme used therefor

ABSTRACT

A method for producing β-hydroxy amino acid and its optically-active isomer is provided. The β-hydroxy amino acid is produced by reacting a predetermined D-α-amino acid and 5,10-methylene tetrahydrofolic acid in the presence of an enzyme derived from a microorganism belonging to the genera  Paracoccus, Aminobacter , or  Ensifer.

This application claims priority under 35 U.S.C. §119(a) to JP2005-148659, filed in Japan on May 20, 2005, the entirety of which isincorporated by reference. The Sequence Listing on Compact Disk filedherewith is also hereby incorporated by reference in its entirety (FileName: US-288 Seq List; File Size: 32 KB; Date Created: May 22, 2006).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing β-hydroxy aminoacid and in particular, to a method for producing the β-hydroxy aminoacid using a novel enzyme.

2. Brief Description of the Related Art

Amino acids such as β-hydroxy amino acid and amino acids having opticalactivity at an α-position are expected to be used as intermediates forpharmaceuticals. Examples of methods for producing optically-activeα-alkyl serine derivatives which are optically-active amino acidderivatives having two different substituents at the α-position, andsalts thereof, include the following methods:

1) asymmetric alkylation of an optically-active oxazolidine compoundobtained from the optically-active serine derivative and pivalaldehyde(Seebach et al., Helvetica Chimica Acta, 1987, 70:1194-1216);

2) asymmetric aldol reaction of α-isocyano carboxylic acid ester andparaformaldehyde with an optically-active metal catalyst (Yoshihiko etal., Tetrahedron Letters, 1988, 29:235-238);

3) asymmetric alkylation of optically-active β-lactam compounds obtainedfrom an optically active oxazolidine chromium carbene complex and anoxazine compound (Colson et al., Journal of Organic Chemistry, 1993,58:5918-5924);

4) asymmetric ring-opening reaction of an optically-active aziridinecompound (Wipf et al., Tetrahedron Letters, 1995, 36:3639-3642) 5)asymmetric alkylation of an optically-active pyrazinone compoundobtained from an optically-active valine derivative and anoptically-active alanine derivative (Najera et al., European Journal ofOrganic Chemistry, 2000, 2809-2820); and

6) Sharpless asymmetric dihydroxylation of a 2-methyl-2-propenoic acidderivative followed by introduction of a resulting optically-active diolcompound into an optically-active azido compound for reduction (Avenozaet al., Tetrahedron Asymmetry, 2001, 12:949-957).

α-Methyl-L-serine is one of the promising substances which may be usedas an intermediate of a medicament. In one of the known methods forproducing α-methyl-L-serine by means of an enzymatic reaction, D-alanineand 5,10-methylenetetrahydrofolic acid are used as the materials, and2-methyl serine hydroxymethyl transferase (EC 2.1.2.7) is used as theenzyme. However, this method utilizes an enzyme derived from amicroorganism belonging to genus Pseudomonas, and requires the additionof expensive α-methyl-serine in order to produce an enzyme in acultivation medium (Wilson et al., J. Biol. Chem 237:3171-3179). Inaddition, utilizing the enzyme derived from the microorganism belongingto genus Pseudomonas, α-methyl-L-serine is obtained from 4 mmol ofmaterial (D-Ala) with a yield of as low as 11%, which does not satisfythe requirements for practical use.

SUMMARY OF THE INVENTION

As mentioned above, many studies have been conducted on a wide varietyof methods for producing optically-active amino acids. Nevertheless, asimpler, more effective, and cost-efficient method for producing avariety of optically-active amino acids and β-hydroxy amino acid isdesirable. The object of the present invention is to provide a newsimpler method for producing β-hydroxy amino acid and optically-activeβ-hydroxy amino acid, as well as an enzyme which may be used in themethod.

A novel method has been developed for producing a β-hydroxy amino acid,and a new protein has been found which catalyzes the reaction in areaction system where 5,10-methylenetetrahydrofolic acid and/or apredetermined aldehyde are involved, and using a D-amino acid as astarting material. It has also been determined that this protein can beused to conveniently produce a β-hydroxy amino acid. In addition, it hasalso been determined that production with this protein results inselective production of an L-amino acid if the product is an amino acidhaving optical activity. The present invention provides a method forproducing a β-hydroxy amino acid and an enzyme used in the method, asmentioned below.

It is an object of the present invention to provide a method forproducing a β-hydroxy amino acid of formula (III):

comprising reacting a D-α-amino acid of formula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

in the presence of an enzyme isolated from a microorganism belonging toa genus selected from the group consisting of Paracoccus, Aminobacter,and Ensifer, and

wherein R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, and

wherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical to any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical to anyof the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and

wherein R¹, R², and R³ may be either linear or branched, and may have asubstituent.

It is a further object of the present invention to provide the methoddescribed above, wherein said D-α-amino acid is D-α-alanine and saidβ-hydroxy amino acid is α-methyl-L-serine.

It is even a further object of the present invention to provide a methodfor producing β-hydroxy amino acid of formula (III):

comprising reacting a D-α-amino acid of formula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

in the presence of a protein selected from the group consisting of:(A) a protein comprising the amino acid sequence of SEQ ID NO: 5;(B) a variant protein of the amino acid sequence of SEQ ID NO: 5, whichis able to catalyze the reaction to produce the β-hydroxy amino acid offormula (III);(C) a protein comprising the amino acid sequence of SEQ ID NO: 11;(D) a variant protein of the amino acid sequence of SEQ ID NO: 11, whichis able to catalyze the reaction to produce the β-hydroxy amino acid offormula (III);(E) a protein comprising the amino acid sequence of SEQ ID NO: 16; and(F) a variant protein of the amino acid sequence of SEQ ID NO: 16, whichis able to catalyze the reaction to produce the β-hydroxy amino acid offormula (III), and

wherein, R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, and

wherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical to any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical to anyof the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and

wherein R¹, R², and R³ may be either linear or branched, and may have asubstituent.

It is even a further object of the present invention to provide themethod as described above, wherein said D-α-amino acid is D-α-alanineand said β-hydroxy amino acid is α-methyl-L-serine.

It is even a further object of the present invention to provide aprotein isolated from a microorganism belonging to a genus selected fromthe group consisting of Paracoccus, Aminobacter, and Ensifer, andwherein said protein is able to catalyze the reaction of a D-α-aminoacid of formula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

to produce a β-hydroxy amino acid of formula (III):

wherein R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, and

wherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical to any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical to anyof the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and

wherein R¹, R², and R³ may be either linear or branched, and may have asubstituent.

It is even a further object of the present invention to provide aprotein which is able to catalyze the reaction of a D-α-amino acid offormula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

to produce a β-hydroxy amino acid of formula (III):

wherein R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, and

wherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical to any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical to anyof the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and

wherein R¹, R², and R³ may be either linear or branched, and may have asubstituent, and

wherein said protein is selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 5, or avariant protein thereof;

(B) a protein comprising the amino acid sequence of SEQ ID NO: 11, or avariant protein thereof;

(C) a protein comprising the amino acid sequence of SEQ ID NO: 16, or avariant protein thereof.

It is even a further object of the present invention to provide apolynucleotide encoding the protein as described above.

It is even a further object of the present invention to provide apolynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4;

(b) a polynucleotide which hybridizes with a nucleotide sequencecomplementary to that of SEQ ID NO: 4 under stringent conditions, andwhich encodes a protein which is able to catalyze the reaction ofD-α-amino acid of formula (I) with 5,10-methylenetetrahydrofolic acidand/or an aldehyde of formula (II) to produce β-hydroxy amino acid offormula (III);

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:10;

(d) a polynucleotide which hybridizes with a nucleotide sequencecomplementary to that of SEQ ID NO: 10 under stringent conditions, andwhich encodes a protein which is able to catalyze the reaction ofD-α-amino acid of formula (I) with 5,10-methylenetetrahydrofolic acidand/or an aldehyde of formula (II) to produce β-hydroxy amino acid offormula (III);

(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;

(f) a polynucleotide which hybridizes with a nucleotide sequencecomplementary to that of SEQ ID NO: 15 under stringent conditions, andwhich encodes a protein which is able to catalyze the reaction ofD-α-amino acid of formula (I) with 5,10-methylenetetrahydrofolic acidand/or an aldehyde of formula (II) to produce β-hydroxy amino acid offormula (III); andwherein formula (I) is:

wherein formula (II) is:

wherein formula (III) is:

wherein, R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, and

wherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical with any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical withany of the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and

wherein R¹, R², and R³ may be either linear or branched and may have asubstituent.

It is even a further object of the present invention to provide arecombinant polynucleotide having the polynucleotide as described aboveincorporated therein.

It is even a further object of the present invention to provide atransformant having the polynucleotide as described above incorporatedtherein.

It is even a further object of the present invention to provide arecombinant polynucleotide having the polynucleotide as described aboveincorporated therein.

It is even a further object of the present invention to provide atransformant having the polynucleotide according to claim 11incorporated therein.

It is even a further object of the present invention to provide themethod as described above, wherein said variant protein of the aminoacid sequence of SEQ ID NO. 5 is 90% homologous to SEQ ID NO. 5, saidvariant protein of the amino acid sequence of SEQ ID NO. 11 is 90%homologous to SEQ ID NO. 11, and said variant protein of the amino acidsequence of SEQ ID NO. 16 is 90% homologous to SEQ ID NO. 16.

It is even a further object of the present invention to provide theprotein as described above, wherein said variant protein in (A) is 90%homologous to SEQ ID NO. 5, said variant protein of (B) is 90%homologous to SEQ ID NO. 11, and said variant protein of (C) is 90%homologous to SEQ ID NO. 16.

The present invention allows a β-hydroxy amino acid to be produced bysimple procedures.

In the production of an optically active β-hydroxy amino acids, thepresent invention allows selective production of an L-amino acid. Thus,the present invention provides an efficient method for producing theL-amino acid. Furthermore, the present invention may achieve theproduction of the recombinant and transformant of the novel enzyme,leading to low-cost, large-scale production of amino acids.

The above and other objects, features, advantages and technical andindustrial significance of this invention will be better understood byreading the following detailed description of the presently preferredembodiments of the invention, when considered in connection with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme showing the reaction system according to oneembodiment of the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The embodiments according to the present invention will be describedhereinbelow with reference to the best mode of carrying out theinvention.

It should be noted that various types of genetic engineering approachesare described in many standard experimental manuals, such as MolecularCloning: A Laboratory Manual, 3^(rd) edition, Cold Spring Harbor press(2001, Jan. 15), Saibo Kogaku Handbook (Cellular Engineering Handbook),Toshio KUROKI et al., Yodosya (1992), and Shin Idenshi Kogaku Handbook(New Gene Engineering Handbook), 3^(rd) edition, Matsumura et al.,Yodosya (1999), and by reference to these manuals, a person skilled inthe art may easily use these approaches.

In the specification, SEQ ID NOs. refers to the sequence numbers in asequence listing unless otherwise stated. In the specification, anenzyme is a protein which is able to catalyze a chemical reaction.

In the method of the present invention for producing the β-hydroxy aminoacid, a D-α-amino acid of formula (I), and 5,10-methylenetetrahydrofolicacid and/or an aldehyde of formula (II) are reacted. In thespecification and the accompanying drawing, tetrahydrofolic acid may besimply referred to as THF. Similarly, 5,10-methylenetetrahydrofolic acidmay be simply referred to as 5,10-methylene THF. 5,10-methylene THFand/or the aldehyde of formula (II) may be used in combination or alone.

Specific examples of R¹ and/or R² may include the following:

Examples of the alkyl group with 1 to 6 carbon atoms may include amethyl group, an ethyl group, a n-propyl group, an isopropyl group, an-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group,a n-pentyl group, an isopentyl group, a neo-pentyl group, a n-hexylgroup, and an isohexyl group.

Examples of the aryl group with 6 to 14 carbon atoms may include aphenyl group, a tolyl group, a xylyl group, a biphenyl group, a naphthylgroup, an anthryl group, and a phenanthryl group.

Examples of the cycloalkyl group with 3 to 10 carbon atoms may include acyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexylgroup, a cycloheptanyl group, a cyclooctanyl group, a cyclononanylgroup, and a cyclodecanyl group.

Examples of the aralkyl group with 7 to 19 carbon atoms may includephenylalkyl groups such as a benzyl group, a benzhydryl group, aphenethyl group and a trityl group, a cinnamyl group, a stylyl group,and a naphthylalkyl group.

Examples of the alkoxyalkyl group with 2 to 11 carbon atoms may includean alkyl group with 1 to 10 carbon atoms which has a substituentselected from the group consisting of a methoxy group, an ethoxy group,a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group,a phenoxy group, a heptoxy group, an octoxy group, a nonanoxy group, anddecanoxy group.

R¹ and/or R² may be a group which is identical with any of theaforementioned hydrocarbon groups except for containing a hetero atom inits carbon skeleton. Examples of the hetero atom may include an oxygenatom, a nitrogen atom, and a sulfur atom.

One embodiment of R¹ and/or R² containing the hetero atom in its carbonskeleton may be a heteroring-containing hydrocarbon group. Theheteroring-containing hydrocarbon group is a cyclic hydrocarbon group,wherein a ring of the cyclic moiety contains the hetero atom. Examplesof the heteroring-containing hydrocarbon group may include heterocyclicgroups such as a heteroaryl group with or without aromaticity and may beeither monocyclic or polycyclic group. Specific examples of theheteroring-containing hydrocarbon group may include a furyl group, athienyl group, a pyridyl group, a piperidyl group, a piperidino group, amorpholino group, an indolyl group, an imidazolyl group, and an alkylgroup substituted by any of these heterocyclic groups.

R¹ and/or R² may also be a hydrocarbon group which is identical with anyof the aforementioned groups except for containing an unsaturatedcarbon-carbon bond in its carbon skeleton.

In addition, the aforementioned R¹ and/or R² may be linear or branched.Moreover, R¹ and/or R² may be the aforementioned hydrocarbon group whichis substituted by the following group or to which the following group isadded: one or more groups which include a halogen atom, an alkyl groupwith up to 3 carbon atoms, an alkoxyl group with up to 3 carbon atoms, aketo group (═O), a hydroxyl group (—OH), a thiol group (—SH), an aminogroup (—NH₂), an amido group (—CONH₂), an imino group (═NH), and ahydrazino group (—NHNH₂).

Examples of the D-α-amino acid of formula (I) may include alanine,valine, leucine, isoleucine, serine, threonine, cysteine, methionine,asparagine, glutamine, phenylalanine, tyrosine, tryptophan, asparticacid, glutamic acid, lysine, arginine, histidine, 2-amino-n-butyricacid, all of which are of D-α-type, preferably, alanine, serine, and2-amino-n-butyric acid, and more preferably, alanine.

Formula (II) does not include formaldehyde (i.e., wherein R² ishydrogen). However, formaldehyde may be used to generate 5,10-methyleneTHF. 5,10-methylene THF may be easily obtained by reacting formaldehydewith THF. 5,10-methylene THF also reacts with a D-amino acid of formula(I) to produce THF. This means that 5,10-THF and THF may form a cyclicreaction system. According to the method of the present invention, theTHF cyclic reaction system may be used as a secondary reaction system.

In formula (III), R¹ is the same as R¹ in formula (I). In formula (III),R³ may be hydrogen, an alkyl group with 1 to 6 carbon atoms, an arylgroup with 6 to 14 carbon atoms, a cycloalkyl group with 3 to 10 carbonatoms, an aralkyl group with 7 to 19 carbon atoms, an alkoxyalkyl groupwith 2 to 11 carbon atoms, a group identical to any of theaforementioned groups except for containing a hetero atom in the carbonskeleton thereof, and a group identical to any of the aforementionedgroups except for containing a carbon-carbon unsaturated bond in thecarbon skeleton thereof, wherein these groups may be either linear orbranched and may have a substituent. Specific examples of thehydrocarbon groups other than hydrogen are the same as those in theaforementioned examples for R¹ and R².

A preferable embodiment of the present invention may be a reactionsystem in which D-α-alanine reacts with 5,10-methylene THF to produceα-methyl-L-serine. FIG. 1 shows a specific example of the reactionsystem.

As shown in FIG. 1, THF reacts with formaldehyde to produce5,10-methylene THF. 5,10-methylene THF is reacted with D-α-alanine inthe presence of a predetermined enzyme. Through the reaction, D-α-methylserine and THF are produced. THF may be reused as a material forsupplying 5,10-methylene THF. In the embodiment where formaldehyde isused to reproduce 5,10-methylene THF, it is preferable that a slightamount of formaldehyde is sequentially added to the reaction system.Since formaldehyde has a high reactivity, the sequential additionthereof to keep pace with the consumption of 5,10-methylene THF mayresult in suppression of by-product production.

Moreover, as shown in the example in FIG. 1, the method of the presentinvention for producing β-hydroxy amino acid is suitable forpreferentially producing the L-isomer of the amino acid when using apredetermined enzyme. The phrase “for preferentially producing theL-isomer” means that the ratio of the L-isomer of the resultingβ-hydroxy amino acid is higher than that of the D-amino acid. The ratioof L-isomer is preferably 70% or more, further preferably 80% or more,and still further preferably 90% or more. The ratio of the L-isomer inthe serine derivative may be calculated by the expression([L-isomer]/([D-isomer]+[L-isomer]))*100.

The reaction temperature is preferably 10 to 50° C. and more preferably20 to 40° C. The pH value for the reaction system is preferably 5 to 9and more preferably 6 to 8.

According to the method of the present invention, 5,10-methylene THFand/or the aldehyde of formula (II) is reacted with D-α-amino acid inthe presence of a predetermined enzyme. The enzyme which catalyzes thereaction may be obtained from a microorganism belonging to generaParacoccus, Aminobacter, or Ensifer. More specific examples of thesemicroorganisms may include Paracoccus sp., Aminobactor sp., and Ensifersp. and preferably Paracoccus sp. FERM BP-10604, Aminobactor sp. FERMBP-10605, and Ensifer sp. FERM BP-10606.

The strains having a FERM number assigned are deposited strains asmentioned below and therefore, may be available by referencing to itsassociated number and the following procedure. These strains were eachconverted into an International Deposit under the provisions of theBudapest Treaty on May 11, 2006.

(1) Name: Paracoccus sp. AJ110402

Deposit number: FERM BP-10604

Depositary authority: International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology

Address: Chuoh No. 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan

Deposit date: Mar. 8, 2005

(2) Name: Aminobactor sp. AJ110403

Deposit number: FERM BP-10605

Depositary authority: International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology

Address: Chuoh No. 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan

Deposit date: Mar. 8, 2005

(3) Name: Ensifer. sp. AJ110404

Deposit number: FERM BP-10606

Depositary authority: International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology

Address: Chuoh No. 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan

Deposit date: Mar. 8, 2005

More specifically, examples of the enzymes used in the reaction forproducing the β-hydroxy amino acid in the present invention may includethe following proteins:

(A) a protein having an amino acid sequence of SEQ ID NO: 5;

(B) a variant of the protein having the amino acid sequence of SEQ IDNO: 5, which is able to catalyze the reaction to produce the β-hydroxyamino acid of formula (III);

(C) a protein having an amino acid sequence of SEQ ID NO: 11;

(D) a variant of the protein having the amino acid sequence of SEQ IDNO: 11, which is able to catalyze the reaction to produce the β-hydroxyamino acid of formula (III);

(E) a protein having an amino acid sequence of SEQ ID NO: 16;

(F) a variant of the protein having the amino acid sequence of SEQ IDNO: 16, which is able to catalyze the reaction to produce the β-hydroxyamino acid of formula (III).

The use of any of the aforementioned proteins may achieve efficientproduction of the β-hydroxy amino acid. According to the method of thepresent invention, among the β-hydroxy amino acids, the L-amino acidform which has an asymmetric carbon in the α-position may be producedwith high selectivity. In particular, in the system where D-α-alaninereacts with 5,10-methylene THF, α-methyl-L-serine only may besubstantially produced, which leads to efficient production of theoptically-active amino acid.

The protein having the amino acid sequence of SEQ ID NO: 5 may beisolated from the Paracoccus sp. FERM BP-10604 strain. The proteinhaving the amino acid sequence of SEQ ID NO: 11 may be isolated from theAminobactor sp. BP-10605 strain. The protein having the amino acidsequence of SEQ ID NO: 16 may be isolated from Ensifer sp. FERM BP-10606strain.

As mentioned above, according to the method of the present invention,proteins which are substantially the same as the proteins (A), (C), and(E), for example, variant proteins, may also be used. For example,protein (B) is a variant of, or substantially the same as, protein (A).A variant protein may have one or more mutations in the amino acidsequence including substitutions, deletions, insertions, additions, andinversions within the sequence. The number of mutations may be one ormore, and may vary depending on the position of the amino acid residueto be mutated in the protein structure and type of the amino acidresidue, and may be a number which does not substantially affect theprotein structure and activity. Specifically, the number of mutationsmay be 1 to 50, preferably 1 to 30, and more preferably 1 to 10.However, the variant protein (B) may desirably have an activity which isapproximately half or more, preferably 80% or more, more preferably 90%or more, and still more preferably 95% or more of the enzyme activitycompared with protein (A) under conditions of 30° C., pH6.5 to 8.0.

The mutations in the amino acid sequence of protein variant (B) may beachieved by alternating the nucleotide sequence so that the amino acidat the specific site of the gene encoding the protein is substituted,deleted, inserted, or added using, e.g., the site-specific mutagenicmethod. Alternatively, the polynucleotide having the nucleotide sequencealtered as mentioned above may be obtained through the knownconventional mutation process. The mutation process may include an invitro treatment of the DNA encoding protein (A) with hydroxyamine or thelike, and a method in which a microorganism belonging to genusEscherichia which carries DNA encoding protein (A) is treated by meansof UV irradiation or with a mutagenic agents commonly used forartificial mutation such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG)and nitrous acid.

The aforementioned mutations may also include naturally-occurringmutations, such as differences between species or between strains of amicroorganism. By expressing the DNA having the mutation(s) mentionedabove in appropriate cells and examining the enzyme activity of theexpressed products, the DNA encoding the protein which is a variant of,or substantially the same protein as, protein (A) may be obtained.

Like the relationship between proteins (A) and (B), protein (D) is anexample of a protein variant which is substantially the same as protein(C), and protein (F) is an example of protein variant which issubstantially the same as protein (E).

Other examples of protein variants which are substantially the same asproteins (A), (B), and (C) may be proteins which have an amino acidsequence resulting in homology of preferably 70% or more, morepreferably 80% or more, and still more preferably 90%, and mostpreferably 95% or more with respect to proteins (A), (B) and (C). In thepresent specification, the homology of the amino acid sequence may beobtained by calculating a matching count percentage over the full-lengthof the polypeptide coded into ORF, using GENETYX software Ver7.0.9(Genetics) with Unit Size to Compare=2, or by its equivalent calculationmethod.

The present invention also provides polynucleotides encoding theaforementioned proteins. Due to codon degeneracy, a certain amino acidsequence may be defined by more than one nucleotide sequence. That is,the polynucleotide of the present invention includes polynucleotideshaving nucleotide sequences which encode any of the aforementionedproteins (A), (B), (C), (D), (E), and (F).

Specifically, examples of the polynucleotide of the present inventionmay include the following polynucleotides:

(a) a polynucleotide having a nucleotide sequence of SEQ ID NO: 4;

(c) a polynucleotide having a nucleotide sequence of SEQ ID NO: 10; and

(e) a polynucleotide having a nucleotide sequence of SEQ ID NO: 15.

The polynucleotide of (a) encodes the protein (A), and may be isolatedfrom the Paracoccus sp. FERM BP-10604 strain. The polynucleotide (c)encodes the protein (C), and may be isolated from the Aminobacter sp.FERM BP-10605 strain. The polynucleotide (e) encodes the protein (E),and may be isolated from the Ensifer sp. FERM BP-10606 strain.

Taking the polynucleotide (a) as an example, a method for isolating thepolynucleotides will be described. The DNA having the nucleotidesequence of SEQ ID NO: 4 may be obtained from a chromosomal DNA ofParacoccus sp. or a DNA library by PCR (polymerase chain reaction, seeWhite, T. J. et al; Trends Genet., 5,185 (1989)) or hybridization. Theprimer used for PCR may be designed based on, for example, the internalamino acid sequence of a purified protein which is able to catalyze thereaction involved in the method of the present invention. Alternatively,the primer or the probe for hybridization may be designed based on thenucleotide sequence of SEQ ID NO: 4, and the DNA may be isolated usingthe probe. A combination of a primer having a sequence corresponding toa 5′ non-translation domain and another primer having a sequencecorresponding to 3′ non-translation domain, between which lies a codingdomain, may be used for the primer for PCR to amplify the full-length ofthe protein coding domain.

The primer may be synthesized in the usual manner, for example, by thephosphoramidite method (see Tetrahedron Letters (1981), 22, 1859) usingDNA synthesizing equipment Model 380B (Applied Biosystems). The PCRprocess may be performed using, for example, Gene Amp PCR System 9600(PERKIN ELMER) and TaKaRa LA PCR in vitro Cloning Kit (TaKaRa Bio)according to the method specified by the the manufacturer.

The polynucleotides which are substantially the same as theaforementioned polynucleotides (a), (c), and (e) are also included inthe polynucleotide of the present invention. The polynucleotides (b),(d) and (f) described below may be enumerated as examples of thepolynucleotide which are substantially the same as the polynucleotides(a), (c) and (e), respectively.

Polynucleotide (b) is able to hybridize with a nucleotide sequencecomplementary to that of SEQ ID NO: 4 under stringent conditions, andencodes a protein which is able to catalyze the reaction of a D-α-aminoacid of formula (I) with 5,10-methylene THF and/or an aldehyde offormula (II) to produce β-hydroxy amino acid of formula (III);

Polynucleotide (d) is able to hybridize with a nucleotide sequencecomplementary to that of SEQ ID NO: 10 under stringent conditions, andencodes a protein which is able to catalyze the reaction of a D-α-aminoacid of formula (I) with 5,10-methylene THF and/or an aldehyde offormula (II) to produce β-hydroxy amino acid of formula (III);

Polynucleotide (f) is able to hybridize with a nucleotide sequencecomplementary to that of SEQ ID NO: 15 under stringent conditions, andencodes a protein which is able to catalyze the reaction of a D-α-aminoacid of formula (I) with 5,10-methylene THF and/or an aldehyde offormula (II) to produce β-hydroxy amino acid of formula (III).

For the polynucleotide to be hybridized, a probe, for example, may beused. In each case, the probe may be prepared in the usual manner basedon the nucleotide sequences of SEQ ID Nos. 4, 10, and 15. The objectivepolynucleotide may be isolated by picking out the nucleotide to behybridized using the probe in the usual manner. The DNA probe, forexample, may be prepared by amplifying the nucleotide sequences clonedinto plasmid or a phage vector, cutting out the desired nucleotidesequence for the probe by a restriction enzyme, and then extracting thesequence. The portion to be cut out may be adjusted according to theobjective DNA. Once the polynucleotide which is substantially the samehas been detected, the polynucleotide may be amplified in the usualmanner such as PCR.

The “stringent conditions” mean conditions under which a so-calledspecific hybrid is formed but a nonspecific hybrid is not formed.Although it is difficult to clearly define the condition in terms ofnumerical values, an example of such conditions may be those under whichthe DNAs having high homology, for example, 50% or more, preferably 70%or more, more preferably 80% or more, further preferably 90% or more,and still further preferably 95% or more, are hybridized while the DNAshaving lower homology are not hybridized. The homology (%) of thenucleotide sequences is represented by numeric values obtained bypercentage calculation over the full-length of ORF of each gene(including a stop codon) using GENETYX software Ver7.0.9 (Genetics) withUnit Size to Compare=6, pick up location=1. As another example,stringent conditions may be those of ordinary washing conditions inSouthern hybridization, under which the DNAs are hybridized at 60° C.and salt concentration of 1×SSC, 0.1% SDS, and preferably 0.1×SSC, 0.1%SDS. The genes hybridized under such conditions may include a genecontaining a stop codon or a gene without activity due to a mutation inthe activity center region. However, these may be easily screened off byinserting the obtained genes in a commercially-available expressionvector, expressing the genes in an appropriate host, and determining theenzyme activity of the expressed product by a method described later.

As mentioned above, in the case of the aforementioned polynucleotide(b), the protein encoded thereby may desirably have an activity ofapproximately half or more, preferably 80% or more, and more preferably90% or more of the activity of protein (A), which is encoded by thenucleotide sequence of SEQ ID NO: 4 under conditions of 30° C., pH8.0.Similarly, in the case of the aforementioned polynucleotide (d), theprotein encoded thereby may desirably have the activity to the sameextent as the above with respect to the protein (C). In the case of theaforementioned polynucleotide (f), the protein encoded thereby maydesirably have the activity to the same extent as the above with respectto the protein (E).

According to the method of the present invention, the enzyme may be usedin any form as long as it is capable of catalyzing the aforementionedreaction in the reaction system. Examples of the specific forms thereofmay include a cultured product of enzyme-producing microorganism, cellsof the microorganism separated from the cultured product, and aprocessed cell product. The cultured product of the microorganism is aproduct obtained by culturing the microorganism. More specifically, thecultured product is a mixture containing the cells of themicroorganisms, the cultivation medium used for culturing themicroorganism, and the substances produced by the culturedmicroorganism. The cells of the microorganisms may be washed beforeusing as the washed cells. The processed cell product may be disrupted,lysed, and/or freeze-dried cells, as well as a crude-purified proteinthat is collected from the processed cells, and a purified protein thatis further purified. As for the purified proteins, a partially-purifiedprotein which is obtained by a variety of types of purification methodsmay be used. Alternatively, a fixed protein which is fixed by a covalentbond method, an adsorption method, or an entrapment method may be used.Depending on the employed microorganism, a part of the cells may belysed during cultivation. In this case, the supernatant of thecultivation medium may also be used as an enzyme-containing substance.

Now, the method for producing the proteins of the present invention andthe method for preparing the recombinants and transformants used inproducing the proteins will be described hereinbelow using theaforementioned protein (A) as an example. The methods which will bedescribed below are also applicable to other proteins.

The transformant which expresses the aforementioned protein (A) may beprepared using a recombinant polynucleotide which contains thepolynucleotide having the aforementioned nucleotide sequence (a)incorporated therein. For example, the transformant may be obtained bypreparing a recombinant DNA containing the DNA having the nucleotidesequence of SEQ ID NO: 4, and then introducing the resulting recombinantDNA into an appropriate host. Examples of the host for expressing theprotein identified by the DNA having the nucleotide sequence of SEQ IDNO: 4 may include a variety of prokaryotic cells, includingmicroorganisms belonging to genus Escherichia such as Escherichia coli,microorganisms belonging to genus Corynebacterium, Bacillus subtilis,and a variety of eukaryotic cells including Saccharomyces cerevisiae,Pichia stipitis, and Aspergillus oryzae. Using the host which may beeasily handled without any expensive components upon cultivation,β-hydroxy amino acid may be easily produced on a large scale.

The recombinant DNA for introducing the DNA having the nucleotidesequence of SEQ ID NO: 4 into a host may be prepared by inserting theDNA into a vector suitable for the type of the host so that the insertedDNA can express the protein encoded thereby. If the promoter inherentlyexists with the gene encoding the aforementioned enzyme derived orisolated from, e.g., Paracoccus sp., Aminobacter sp., and Ensifer sp.are capable of functioning in the host cells, such a promoter may beused as the promoter for facilitating the expression of the proteins.Alternatively, if necessary, any other promoter which can function inthe host may be coupled to the DNA of SEQ ID NO: 4 or the like so thatthe proteins are expressed under the control of the promoter.

Examples of methods for transforming the recombinant DNA to introducethe recombinant DNA into the host cell may include the D. M. Morrison'smethod (Methods in Enzymology 68, 326 (1979)) and a method for improvingthe permeability of the DNA by treating a recipient cell with calciumchloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

In the case of producing an objective protein on a large scale using therecombinant DNA technology, one of the preferable embodiments may be theformation of an inclusion body of the protein. The inclusion body isconfigured by aggregation of the protein in the protein-producingtransformant. The advantages of this expression production method may beprotection of the objective protein from digestion due to protease inthe microbial cells, and ready purification of the objective proteinthat may be performed by disruption of the microbial cells and followingcentrifugation. To obtain the active protein from the protein inclusionbody, a series of manipulations such as solubilization and activityregeneration is required, and thus, the manipulations are morecomplicated than those used when directly producing the active protein.However, when a protein which affects microbial cell growth is producedon a large scale in the microbial cells, effects thereof may be avoidedby accumulating the protein as an inactive inclusion body in themicrobial cells.

Examples of the methods for producing the objective protein on a largescale as an inclusion body may include methods of expressing the proteinalone under the control of a strong promoter, as well as methods ofexpressing the objective protein as a fusion protein with a proteinknown to be expressed in a large amount.

As the host to be transformed, any strain commonly used in expressingheterogenes may be used. Suitable examples thereof may include theEscherichia coli JM109, DH5α, HB101, and BL21 strains, which aresubspecies of the Escherichia coli K12 strain. The method fortransforming the host and the method for selecting out the transformantsare described in Molecular Cloning: A Laboratory Manual, 3rd edition,Cold Spring Harbor press (2001/01/15). An example of the method forpreparing the transformed Escherichia coli strain and producing apredetermined enzyme using the transformed strain will be specificallydescribed hereinbelow.

As the promoter for expressing the DNA encoding the mutant protein, thepromoters typically used for producing xenogenic proteins in E. coli maybe used, and examples thereof may include strong promoters such as T7promoter, lac promoter, trp promoter, trc promoter, tac promoter, and PRpromoter and PL promoter of lambda phage. As the vector, pUC19, pUC18,pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118,pMW219, pMW218, pACYC177, pACYC184, and derivatives thereof may be used.Other vectors of phage DNA may also be used. In addition, expressionvectors which contain a promoter and can express the inserted DNAsequence may also be used.

In order to produce the mutant protein as a fusion protein inclusionbody, a fusion protein gene is produced by linking a gene encodinganother protein, preferably a hydrophilic peptide, upstream ordownstream of the mutant protein gene. Such a gene encoding anotherprotein may be those which increase the amount of the accumulated fusionprotein and enhance solubility of the fusion protein after denaturationand regeneration steps. Examples of candidates thereof may include T7gene 10, β-galactosidase gene, dehydrofolic acid reductase gene,interferon γ gene, interleukin-2 gene and prochymosin gene.

Such a gene may be ligated to the gene encoding the mutant protein sothat reading frames of codons are matched. This may be effected byligating at an appropriate restriction enzyme site or using a syntheticDNA having an appropriate sequence.

In some cases, it is preferable to ligate a terminator, i.e. thetranscription termination sequence, downstream of the fusion protein inorder to increase the production amount. Examples of this terminator mayinclude T7 terminator, fd phage terminator, T4 terminator, tetracyclineresistant gene terminator, and E. coli trpA gene terminator.

The vector for introducing the gene encoding the mutant protein or thefusion protein of the mutant protein with the other protein into E. colimay preferably be of a so-called multicopy type. Examples thereof mayinclude plasmids having a replication origin derived from ColE1, such aspUC based plasmids, pBR322 based plasmids or derivatives thereof. Asused herein, the “derivative” means the plasmid modified by thesubstitution, deletion, insertion, addition and/or inversion of thenucleotides. “Modified” referred to herein includes the modification bymutagenesis with the mutagen or UV irradiation and natural mutation.

In order to select the transformants, it is preferable to employ avector having a marker such as an ampicillin resistant gene. As such aplasmid, expression vectors having the strong promoter are commerciallyavailable (pUC series: Takara Shuzo Co., Ltd., pPROK series andpKK233-2: Clontech, etc.).

A DNA fragment where the promoter, the gene encoding the protein havingthe objective activity or the fusion protein of the objective proteinwith the other protein, and in some cases the terminator are ligatedsequentially, is then ligated to the vector DNA to obtain a recombinantDNA.

The resulting recombinant DNA is used to transform Escherichia coli andthen the transformed Escherichia coli is cultured, to express andproduce the predetermined protein or its fused protein.

In the case of expressing the fusion protein, the fusion protein may becomposed so as to be able to cleave out the objective enzyme therefromusing a restriction protease which recognizes a sequence of bloodcoagulation factor Xa, kallikrein or the like which is not present inthe objective enzyme.

As production media, media such as M9-casamino acid medium and LB mediumwhich are typically used for cultivation of E. coli may be used. Theconditions for cultivation and a production induction may beappropriately selected depending on types of the marker and the promoterof the vector and the host used.

The following methods are available for recovering the objective proteinor the fusion protein containing the objective protein with the otherprotein. If the objective protein or the fusion protein thereof issolubilized in the microbial cells, the cells may be collected and thendisrupted or lysed to thereby obtain a crude enzyme solution for use. Ifnecessary, the crude solution may be purified using techniques such asordinary precipitation, filtration and column chromatography, to obtainthe purified objective protein or the fusion protein. In this case, thepurification may be performed using an antibody against the mutantprotein or the fusion protein. In the case where the protein inclusionbody is formed, it may be solubilized with a denaturant, and then thedenaturant may be removed by means of dialysis or the like to obtain theobjective protein.

EXAMPLES

The present invention will be described in more detail with reference tothe following non-limiting examples.

Example 1 Detection of 2-methylserine hydroxylmethyl transferaseActivity

In a nutrient broth agar medium (Difco), microorganisms listed in Table1 were cultured at 30° C. for 24 hours. A platinum loopful of theresulting cells were inoculated into 3 ml of nutrient broth liquidmedium and then cultured at 30° C. for 24 hours, with 120reciprocations/minute. 0.15 ml of the resulting cultured solution wasinoculated into 3 ml of nutrient broth liquid medium containing 0.2%α-methyl-DL-serine and cultured at 30° C. for 24 hours with 120reciprocations/minute.

After cultivation, the cells were centrifuged and then washed twice withan equal volume of 50 mM potassium phosphate buffer (pH7.4) containing0.1 mM pyridoxal phosphoric acid. 50 M potassium phosphate buffer(pH7.4) containing 0.1 mM pyridoxal phosphoric acid was used to preparea total amount (0.3 ml) of cell suspension and then the suspension wasultrasonically disrupted at 4° C. The supernatant obtained bycentrifugation (16,000 g, 10 min.) was dialyzed with 50 mM potassiumphosphate buffer (pH7.4) containing 0.1 mM pyridoxal phosphoric acid toobtain a cell-free extracted solution.

0.05 ml of cell-free extracted solution was added to a reaction solution(1), which has a composition of 50 mM potassium phosphate buffer(pH7.4), 10 mM α-methyl-DL-serine, 0.5 mM tetrahydrofolic acid, 10 mM2-mercaptoethanol, 0.01 mM pyridoxal phosphoric acid, 10 mM sodiumascorbate, 0.4 mM NADP, and 1 U/ml 5,10-methylene tetrahydrofolic aciddehydrogenase. The total amount (0.1 ml) of solution was reacted at 30°C. for 10 minutes. The reaction was stopped with 0.15 ml of 0.6 Nhydrochloric acid. The supernatant obtained by centrifugation (16,000 g,10 minutes) was left to stand at room temperature for 10 minutes.Absorbance (E11) of the supernatant caused by5,10-methenyl-5,6,7,8-tetrahydrofolic acid was measured at 350 nm. As acontrol, another reaction was performed in the same way as the above,except that α-methyl-DL-serine was replaced with water in theaforementioned solution (1), and the absorbance (E10) caused by5,10-methenyl-5,6,7,8-tetrahydrofolic acid in the resulting liquid wasmeasured. Based on the measured absorbance, the alteration in theabsorbance specific to α-methyl-DL-serine (EΔ1=E11-E10) was calculated.The results are shown in Table 1. TABLE 1 Strain EΔ1 Paracoccus 1.35 sp.A13 Aminobacter 2.07 sp. A10 Ensifer sp. 1.25 B30

Example 2 Purification of 2-methyl serine hydroxylmethyl transferaseDerived from the Paracoccus sp. AJ110402 Strain

(1) Preparation of Cell-Free Extracted Solution

Cells of Paracoccus species were cultured in the nutrient broth agarmedium (Difco) at 30° C. for 24 hours. The cultured cells wereinoculated into 50 ml of nutrient broth liquid medium in a 500 mlSakaguchi flask and cultured at 30° C. for 24 hours with 120reciprocations/minute. The resulting cultured solution was inoculatedinto 2 L of liquid medium containing 0.2% α-methyl-DL-serine, and 0.17%yeast nitrogen base w/o amino acid and ammonium sulfate (pH7.0). 50 mleach of the mixture was dispensed into each of the 500 ml Sakaguchiflasks, and then cultured at 30° C. for 22 hours with 120reciprocations/minute. The resulting cells were collected bycentrifugation (8,000 g, 10 minutes) and washed twice with 25 mMpotassium phosphate buffer containing 0.02 mM pyridoxal phosphoric acid(referred to hereinafter as the buffer (I)). Then, 100 ml of cellsuspension was prepared using the buffer (I). The cells wereultrasonically disrupted and centrifuged (18,000 g, 10 minutes), and theresulting supernatant was ultra-centrifuged (200,000 g, 30 minutes). Theresulting supernatant was used as the cell-free extracted solution.

(2) Anion-Exchange Chromatography

The cell-free extracted solution was applied to a ResourceQ column(Amersham Biosciences) which had been previously equilibrated with thebuffer (I), and the enzyme was eluted by a linear concentration gradientof 0-1M sodium chloride. This process was conducted twice by dividingthe cell-free extracted solution into two aliquots.

(3) Hydrophobic Interaction Chromatography

Active fractions of the enzyme obtained in the aforementioned (2) weremixed with the buffer (I) containing an equivalent amount of 2M ammoniumsulfate and then applied to the Phenyl-Sepharose column (AmashamBiosciences) which had been previously equilibrated with the buffer (I)containing 1M ammonium sulfate. Then, the enzyme was eluted by thelinear concentration gradient of 1-0M ammonium sulfate.

(4) Hydroxyapatite Column Chromatography

The active fractions obtained in the aforementioned (3) were dialyzedwith the 2.5 mM potassium phosphate buffer (pH6.8) containing 0.02 mMpyridoxal phosphoric acid and then applied to the CellulofineHAp columns(SEIKAGAKU Corp.) which had been previously equilibrated with the samebuffer. The enzyme was eluted with the 2.5-250 mM potassium phosphatebuffer (pH6.8). The active fractions of the enzyme were used as thepurified enzyme in the following experiments.

The enzyme thus obtained in this way had specific activity of 3.51 U/mg.The enzyme was electrophoresed in SDS-polyacrylamide and the gel wasstained with a Coomassie brilliant blue staining fluid. A homogeneousband appeared at a position of the molecular weight of approximately47,000.

Example 3 Determination of the Amino Acid Sequence of 2-methyl serinehydroxylmethyl transferase Derived From the Paracoccus sp. AJ110402Strain and the Nucleotide Sequence Encoding the Same

50 pmol of the purified enzyme which had been prepared in Example 2 waselectrophoresed in SDS-polyacrylamide, and transferred on a PVDFmembrane. The relevant part was put in a protein sequencer to determine30 amino acids (SEQ ID NO: 1).

Subsequently, 5 μg of genome DNA derived from the Paracoccus sp.AJ110402 strain was cleaved with PstI (50 U) and then ligated to thePstI cassette of TaKaRa LA PCR in vitro Cloning Kit in accordance withthe kit directions. Using the ligated mixture as a template, PCR (94°C.: 30 sec., 47° C.: 2 min., 72° C.: 1 min., 30 cycles) was performedwith a cassette primer C1 and a primer HMT_FW1 (SEQ ID NO: 2). Using thePCR reaction solution as a template, the second PCR (94° C.: 30 sec.,55° C.: 2 min., 72° C.: 1 min., 30 cycles) was performed with a cassetteprimer C2 and a primer HMT_FW2 (SEQ ID NO: 3). Approximately 0.7kb-length fragments, of which amplification had been confirmed, wereligated to pGEM-Teasy (Promega), with which Escherichia coli JM109strain was then transformed. The nucleotide sequence of the plasmidhaving the objective fragment was confirmed with a DNA sequencer(ABI3100). Approximately 1.1 kb-length gene fragment was obtained bytreating the plasmid with EcoRI/PstI. Using the fragment as a probe,chromosomal DNAs were subjected to Southern analysis after treatmentwith various types of restriction enzymes. When the treatment wasperformed with BglII/NruI, a positive signal was confirmed in anapproximately 3.5 kb region.

Subsequently, the chromosomal DNAs were treated with BglII/NruI and thenelectrophoresed in an agarose gel, to purify the approximately 3.5 kbfragment. The fragment was then ligated to the pUC19 BamHI/SmaI site.Using this reaction solution, Escherichia coli JM109 was transformed tocreate a library. The aforementioned probe was used to perform colonyhybridization for obtaining positive colonies. A plasmid was extractedfrom the positive colonies. The plasmid was designated pHMT01. As to theinserted 3475 bp part, the nucleotide sequence thereof was determined.As a result, an ORF (SEQ ID NO: 4) composed of 425 amino acids wasfound. The ORF had the same sequence as the amino acid sequence obtainedfrom N-terminal analysis, which confirms that the objective gene wasobtained. As for the ORF of the gene sequence, a homology search wasconducted. As a result, 55% homology was confirmed between serinehydroxylmethyl transferase derived from Methylobacterium extorquens andthe ORF amino acid sequence.

Example 4 Expression of 2-methylserine hydroxylmethyl transferase GeneDerived from the Paracoccus sp. AJ110402 Strain in Escherichia coli

Using pHMT01 as a template, PCR was performed with a primer PHMT_SD_Eco(SEQ ID NO: 6) and a primer PHMT_ter2_Hind (SEQ ID NO: 7) to amplify a1.2 kb region of 2-methylserine hydroxylmethyl transferase. Theamplified sequence was treated with EcoRI/HindIII, and then ligated topUC18 which had been previously treated with EcoRI/HindIII. With theligated product, Escherichia coli JM109 strain was transformed. Atransformant having plasmid (pUCPHMT01) containing the objective genefragment was thus obtained. The transformant was designatedJM109/pUCPHMT01.

JM109/pUCPHMT01 was pre-cultured in the LB medium containing 100 mg/Lampicillin at 37° C. for 16 hours. 2.5 ml of the pre-cultured solutionwas inoculated into 50 ml of the LB medium containing 100 mg/Lampicillin and cultured at 37° C. One hour after the onset of thecultivation, IPTG was added so that the final concentration thereofreached 1 mM. The mixture was further cultured for four hours. Theresulting cells were collected by centrifugation and washed with the 50mM phosphoric acid buffer (pH7.4) containing 0.1 mM pyridoxal phosphoricacid. A cell suspension was then prepared using the same buffer. Thecells were ultrasonically disrupted and centrifuged (18,000 g, 10 min.,4° C.) to obtain a supernatant. 2-methylserine hydroxylmethyltransferase activity was measured as to the supernatant as the cell-freeextracted solution. The measured value was 3.02 U/mg. Apart from theabove, pUC18 was introduced into JM109 strain to obtain a transformantJM109/pUC18, and a cell-free extracted solution was prepared therefromand the activity was measured in the same manner as described above. Themeasured activity was less than the detection limit.

Example 5 Isolating the 2-methylserine hydroxylmethyl transferase GeneFrom the Aminobactor sp. AJ110403 Strain

Using the genomic DNA prepared from the Aminobactor sp. AJ110403 strainas a template, PCR was performed with mixed primers HMT_MIX_FW1 (SEQ IDNO: 8) and HMT_MIX_RV2 (SEQ ID NO: 9). An amplified fragment of 0.6 kbwas confirmed. Using this PCR product as a probe, the genome DNA of theAminobactor sp. AJ110403 strain was subjected to BamHI treatment andSouthern analysis. As a result, a positive signal appeared in a 3.5kb-length region.

Subsequently, the genomic DNA of the Aminobactor sp. AJ110403 strain wastreated with BamHI and electrophoresed in the agarose gel. A fragment ofapproximately 3.5 kb was purified. The fragment was ligated to thepUC118 BamHI site. Using this reaction solution, Escherichia coli JM109was transformed to create a library. The aforementioned probe was usedto perform colony hybridization for obtaining positive colonies, and aplasmid was extracted from the positive colonies. The plasmid wasdesignated pAHMT01. As for the inserted part in the plasmid, thenucleotide sequence thereof was determined. As a result, existence of anORF composed of 425 amino acids was confirmed (SEQ ID NO: 10).

Using pAHMT01 as a template, PCR was performed with primers A2_Bam (SEQID NO: 12) and A2_ter_Pst (SEQ ID NO: 13). The amplified fragment of 1.2kb obtained by PCR was treated with BamHI/PstI, and then inserted intopUC18 BamHI/PstI site, to obtain pUCAHMT01. Using this plasmid,Escherichia coli JM109 was transformed. The transformant was designatedJM109/pUCAHMT01.

JM109/pUCAHMT01 was pre-cultured in the LB medium containing 100 mg/Lampicillin at 37° C. for 16 hours. 2.5 ml of pre-cultured solution wasinoculated into 50 ml of the LB medium containing 100 mg/L ampicillinand cultured at 37° C. One hour after the onset of the cultivation, IPTGwas added so that the final concentration thereof reached 1 mM. Themixture was further cultured for four hours. The resulting cells werecollected by centrifugation and washed with the 50 mM phosphoric acidbuffer (pH7.4) containing 0.1 mM pyridoxal phosphoric acid. A cellsuspension was then prepared using the same buffer. The cells wereultrasonically disrupted and centrifuged (18,000 g, 10 min., 4° C.) toobtain a supernatant. 2-methylserine hydroxylmethyl transferase activitywas measured as to the supernatant as the cell-free extracted solution.The measured value was 0.27 U/mg. The activity as to the cell-freeextracted solution prepared from JM109/pUC18 in the same manner asdescribed above was less than the detection limit.

Example 6 Isolating 2-methylserine hydroxylmethyl transferase Gene Fromthe Ensifer sp. AJ110404 Strain

Using genomic DNA prepared from the Ensifer sp. AJ110404 strain as atemplate, PCR was performed with mix primers HMT_MIX_FW2 (SEQ ID NO: 14)and HMT_MIX_RV2 (SEQ ID NO: 9). An amplified fragment of 0.6 kb wasconfirmed. Using this PCR product as a probe, the genomic DNA from theEnsifer sp. AJ strain was subjected to EcoRI treatment and Southernanalysis. As a result, a positive signal appeared in a 5 kb-lengthregion.

Subsequently, genomic DNA from the Ensifer sp. AJ110404 strain wastreated with EcoRI and electrophoresed in the agarose gel. A fragment ofapproximately 5 kb was purified. The fragment was ligated to the pUC118EcoRI site. Using this reaction solution, Escherichia coli JM109 wastransformed to create a library. The aforementioned probe was used toperform colony hybridization for obtaining positive colonies, and aplasmid was extracted from the positive colonies. The plasmid wasdesignated pEHMT01. As to the inserted part in the plasmid, thenucleotide sequence thereof was determined. As a result, existence of anORF composed of 425 amino acids was confirmed (SEQ ID NO: 15).

Using pEHMT01 as a template, PCR was performed with primers B_Eco (SEQID NO: 17) and B_ter_Bam (SEQ ID NO: 18). The amplified fragment of 1.2kb obtained by PCR was treated with BamHI/EcoRI, and then inserted intopUC18 BamHI/EcoRI site, to obtain pUCEHMT01. Using this plasmid,Escherichia coli JM109 was transformed. The transformant was designatedJM109/pUCEHMT01.

JM109/pUCEHMT01 was pre-cultured in the LB medium containing 100 mg/Lampicillin at 37° C. for 16 hours. 2.5 ml of pre-cultured solution wasinoculated into 50 ml of the LB medium containing 100 mg/L ampicillinand cultured at 37° C. One hour after the onset of the cultivation, IPTGwas added so that the final concentration thereof reached 1 mM. Themixture was further cultured for four hours. The resulting cells werecollected by centrifugation and washed with the 50 mM phosphoric acidbuffer (pH7.4) containing 0.1 mM pyridoxal phosphoric acid. A cellsuspension was then prepared using the same buffer. The cells wereultrasonically disrupted and centrifuged (18,000 g, 10 min., 4° C.) toobtain a supernatant. 2-methylserine hydroxylmethyl transferase activitywas measured as to the supernatant as the cell-free extracted solution.The measured value was 0.10 U/mg. The activity as to the cell-freeextracted solution prepared from JM109/pUC18 in the same manner asdescribed above was less than the detection limit.

Example 7 Production of α-methyl-L-serine with 2-methylserinehydroxylmethyl transferase Isolated from Paracoccus sp. AJ110402

The purified enzyme solution which was prepared in Example 2 was addedto a composition composed of 100 mM D-alanine, 20 mM formaldehyde, 0.5mM tetrahydrofolic acid, 10 mM sodium ascorbate, 10 mM2-mercaptoethanol, 0.1 mM pyridoxal phosphoric acid, and 50 mMphosphoric acid buffer (pH7.4), so that the final concentration of theenzyme reached 47 μg/ml. The reaction was performed at 30° C. for 16hours. As formaldehyde, the highest quality formaldehyde liquid [codeNo.: 16223-55] from Nakarai Tesque was used.

After the reaction, an equivalent amount of 2 mM aqueous copper sulfatewas added thereto and HPLC was performed using Sumichiral OA-6100(Sumika Chemical Analysis Service, Ltd.)(mobile phase: 1 mM aqueouscopper sulfate, column temperature: 40° C., flow rate: 1 ml/min.,detection: UV215 nm). As a result, 19 mM of α-methyl-L-serine wasdetected but no peak attributed to α-methyl-D-serine was detected.

Example 8 Production of α-methyl-L-serine with Escherichia coliExpressing the 2-methylserine hydroxylmethyl transferase Gene Isolatedfrom Paracoccus sp. AJ110402

Using the method described in Example 4, 400 ml of the cultured liquidof JM109/pUCPHMT01 was prepared. After centrifugation, the cells werewashed with 50 mM phosphoric acid buffer (pH8.0) containing 0.1 mMpyridoxal phosphoric acid. The cells were added to the 100 ml ofreaction solution (150 mM (15 mmol) D-alanine, 0.1 mM pyridoxalphosphoric acid, 0.3 mM tetrahydrofolic acid, 10 mM 2-mercaptoethanol,20 mM phosphoric acid buffer (pH8.0)) and then 50.5 ml of 600 mM aqueousformaldehyde was added thereto over 24 hours at 30° C. while stirring.As formaldehyde, the highest quality formaldehyde liquid from NakaraiTesque [code number: 16223-55] was used.

Under the same conditions as those in Example 7, HPLC analysis wasperformed. As a result, 66.4 mM (9.5 mmol) of α-methyl-L-serine wasdetected in the reaction solution but the amount of α-methyl-D-serineproduction was less than the detection limit.

Example 9 Homology of Proteins

Homology was examined for the amino acid sequences of the enzymesobtained in the aforementioned examples. In calculating the homology ofthe amino acid sequences, Marching count percentage was calculated overthe full-length of the polypeptide chain encoded in ORF using GENETYXsoftware Ver7.0.9 (Genetics) with Unit Size to Compare=2.

Amino acid sequences of the following enzymes derived fromMethylobacterium Extorquens have been deposited as accession No.AAA64456 in GenBank (National Center for Biotechnology Information).Amino acid sequences of the following enzymes derived from Escherichiacoli have been deposited as accession No. AAA23912 in GenBank. TABLE 2Paracoccus Aminobacter sp. sp. Ensifer sp. SEQ ID SEQ ID NO: SEQ ID NO:Methylobacterium NO: 5 11 16 Extorquens E. coli Paracoccus sp. 100 84.780.0 55.5 50.4 SEQ ID NO: 5 Example EC 2.1.2.7 Aminobacter sp. 100 82.856.1 51.9 SEQ ID NO: 11 Example EC 2.1.2.7 Ensifer sp. 100 55.8 49.3 SEQID NO: 16 Example EC 2.1.2.7 Methylobacterium 100 59.8 ExtorquensComparative example EC 2.1.2.1 E. coli 100 Comparative example EC2.1.2.1

INDUSTRIAL APPLICABILITY

The method of the present invention is useful in the industriesinvolving in amino acid production. It is expected that the presentinvention will contribute to the production of various types ofβ-hydroxy amino acid and optically-active amino acid, and specifically,the method may be used in producing, for example, intermediates forpharmaceuticals.

Although the present invention has been described with reference to thepreferred examples, it should be understood that various modificationsand variations can be easily made by those skilled in the art withoutdeparting from the spirit of the invention. Accordingly, the foregoingdisclosure should be interpreted as illustrative only and is not to beinterpreted in a limiting sense. The present invention is limited onlyby the scope of the following claims along with their full scope ofequivalents. Each of the aforementioned documents, including the foreignpriority document, is incorporated by reference herein in its entirety.

1. A method for producing a β-hydroxy amino acid of formula (III):

comprising reacting a D-α-amino acid of formula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

in the presence of an enzyme isolated from a microorganism belonging toa genus selected from the group consisting of Paracoccus, Aminobacter,and Ensifer, and wherein R¹ and R² are each selected from the groupconsisting of an alkyl group with 1 to 6 carbon atoms, an aryl groupwith 6 to 14 carbon atoms, a cycloalkyl group with 3 to 10 carbon atoms,an aralkyl group with 7 to 19 carbon atoms, an alkoxyalkyl group with 2to 11 carbon atoms, a group identical to any of the aforementionedgroups except for containing a hetero atom in the carbon skeletonthereof, and a group identical to any of the aforementioned groupsexcept for containing a carbon-carbon unsaturated bond in the carbonskeleton thereof, and wherein R³ is selected from the group consistingof hydrogen, an alkyl group with 1 to 6 carbon atoms, an aryl group with6 to 14 carbon atoms, a cycloalkyl group with 3 to 10 carbon atoms, anaralkyl group with 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to11 carbon atoms, a group identical to any of the aforementioned groupsexcept for containing a hetero atom in the carbon skeleton thereof, anda group identical to any of the aforementioned groups except forcontaining a carbon-carbon unsaturated bond in the carbon skeletonthereof, and wherein R¹, R², and R³ may be either linear or branched,and may have a substituent.
 2. The method for producing the β-hydroxyamino acid according to claim 1, wherein said D-α-amino acid isD-α-alanine and said β-hydroxy amino acid is α-methyl-L-serine.
 3. Amethod for producing β-hydroxy amino acid of formula (III):

comprising reacting a D-α-amino acid of formula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

in the presence of a protein selected from the group consisting of: (A)a protein comprising the amino acid sequence of SEQ ID NO: 5; (B) avariant protein of the amino acid sequence of SEQ ID NO: 5, which isable to catalyze the reaction to produce the β-hydroxy amino acid offormula (III); (C) a protein comprising the amino acid sequence of SEQID NO: 11; (D) a variant protein of the amino acid sequence of SEQ IDNO: 11, which is able to catalyze the reaction to produce the β-hydroxyamino acid of formula (III); (E) a protein comprising the amino acidsequence of SEQ ID NO: 16; and (F) a variant protein of the amino acidsequence of SEQ ID NO: 16, which is able to catalyze the reaction toproduce the β-hydroxy amino acid of formula (III), and wherein, R¹ andR² are each selected from the group consisting of an alkyl group with 1to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms, a cycloalkylgroup with 3 to 10 carbon atoms, an aralkyl group with 7 to 19 carbonatoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a group identicalto any of the aforementioned groups except for containing a hetero atomin the carbon skeleton thereof, and a group identical to any of theaforementioned groups except for containing a carbon-carbon unsaturatedbond in the carbon skeleton thereof, and wherein R³ is selected from thegroup consisting of hydrogen, an alkyl group with 1 to 6 carbon atoms,an aryl group with 6 to 14 carbon atoms, a cycloalkyl group with 3 to 10carbon atoms, an aralkyl group with 7 to 19 carbon atoms, an alkoxyalkylgroup with 2 to 11 carbon atoms, a group identical to any of theaforementioned groups except for containing a hetero atom in the carbonskeleton thereof, and a group identical to any of the aforementionedgroups except for containing a carbon-carbon unsaturated bond in thecarbon skeleton thereof, and wherein R¹, R², and R³ may be either linearor branched, and may have a substituent.
 4. The method for producing theβ-hydroxy amino acid according to claim 3, wherein said D-α-amino acidis D-α-alanine and said β-hydroxy amino acid is α-methyl-L-serine.
 5. Aprotein isolated from a microorganism belonging to a genus selected fromthe group consisting of Paracoccus, Aminobacter, and Ensifer, andwherein said protein is able to catalyze the reaction of a D-α-aminoacid of formula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

to produce a β-hydroxy amino acid of formula (III):

wherein R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, andwherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical to any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical to anyof the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and wherein R¹, R², andR³ may be either linear or branched, and may have a substituent.
 6. Aprotein which is able to catalyze the reaction of a D-α-amino acid offormula (I):

with 5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula(II):

to produce a β-hydroxy amino acid of formula (III):

wherein R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, andwherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical to any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical to anyof the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and wherein R¹, R², andR³ may be either linear or branched, and may have a substituent, andwherein said protein is selected from the group consisting of: (A) aprotein comprising the amino acid sequence of SEQ ID NO: 5, or a variantprotein thereof; (B) a protein comprising the amino acid sequence of SEQID NO: 11, or a variant protein thereof; (C) a protein comprising theamino acid sequence of SEQ ID NO: 16, or a variant protein thereof.
 7. Apolynucleotide encoding the protein of claim
 6. 8. A polynucleotideselected from the group consisting of: (a) a polynucleotide comprisingthe nucleotide sequence of SEQ ID NO: 4; (b) a polynucleotide whichhybridizes with a nucleotide sequence complementary to that of SEQ IDNO: 4 under stringent conditions, and which encodes a protein which isable to catalyze the reaction of D-α-amino acid of formula (I) with5,10-methylenetetrahydrofolic acid and/or an aldehyde of formula (II) toproduce β-hydroxy amino acid of formula (III); (c) a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 10; (d) apolynucleotide which hybridizes with a nucleotide sequence complementaryto that of SEQ ID NO: 10 under stringent conditions, and which encodes aprotein which is able to catalyze the reaction of D-α-amino acid offormula (I) with 5,10-methylenetetrahydrofolic acid and/or an aldehydeof formula (II) to produce β-hydroxy amino acid of formula (III); (e) apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 15; (f)a polynucleotide which hybridizes with a nucleotide sequencecomplementary to that of SEQ ID NO: 15 under stringent conditions, andwhich encodes a protein which is able to catalyze the reaction ofD-α-amino acid of formula (I) with 5,10-methylenetetrahydrofolic acidand/or an aldehyde of formula (II) to produce β-hydroxy amino acid offormula (III); and wherein formula (I) is:

wherein formula (II) is:

wherein formula (III) is:

wherein, R¹ and R² are each selected from the group consisting of analkyl group with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbonatoms, a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl groupwith 7 to 19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbonatoms, a group identical to any of the aforementioned groups except forcontaining a hetero atom in the carbon skeleton thereof, and a groupidentical to any of the aforementioned groups except for containing acarbon-carbon unsaturated bond in the carbon skeleton thereof, andwherein R³ is selected from the group consisting of hydrogen, an alkylgroup with 1 to 6 carbon atoms, an aryl group with 6 to 14 carbon atoms,a cycloalkyl group with 3 to 10 carbon atoms, an aralkyl group with 7 to19 carbon atoms, an alkoxyalkyl group with 2 to 11 carbon atoms, a groupidentical with any of the aforementioned groups except for containing ahetero atom in the carbon skeleton thereof, and a group identical withany of the aforementioned groups except for containing a carbon-carbonunsaturated bond in the carbon skeleton thereof, and wherein R¹, R², andR³ may be either linear or branched and may have a substituent.
 9. Arecombinant polynucleotide having the polynucleotide according to claim7 incorporated therein.
 10. A transformant having the polynucleotideaccording to claim 9 incorporated therein.
 11. A recombinantpolynucleotide having the polynucleotide according to claim 8incorporated therein.
 12. A transformant having the polynucleotideaccording to claim 11 incorporated therein.
 13. The method of claim 3,wherein said variant protein of the amino acid sequence of SEQ ID NO. 5is 90% homologous to SEQ ID NO. 5, said variant protein of the aminoacid sequence of SEQ ID NO. 11 is 90% homologous to SEQ ID NO. 11, andsaid variant protein of the amino acid sequence of SEQ ID NO. 16 is 90%homologous to SEQ ID NO.
 16. 14. The protein of claim 6, wherein saidvariant protein in (A) is 90% homologous to SEQ ID NO. 5, said variantprotein of (B) is 90% homologous to SEQ ID NO. 11, and said variantprotein of (C) is 90% homologous to SEQ ID NO. 16.